Simple and Rapid LC–MS/MS Methods for Quantifying Catabolites of Antibody-Drug Conjugates with SMCC Linker

Abstract The stability and exposure of toxin-related catabolites in system circulation contributes to the evaluation of the stability, targeted delivery and off-target toxicity for antibody–drug conjugates (ADC) at different stages during drug development. In this study, simple and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for determination catabolites of Mertansine (DM1), MCC-DM1 and Lys-MCC-DM1 in cynomolgus serum have been developed. The serum samples are processed by protein precipitation. The LC–MS/MS methods are applied on a Phenomenex C8 column (50 × 2.0 mm, 5 μm) with gradient elution with water–formic acid 0.1% (A) and acetonitrile-formic acid 0.1% (B) at a flow rate of 0.5 mL/min. The analytical run time is only 4.0 min and the calibration ranges of the standard curve are 0.500–200 ng/mL for DM1, 1.00–500 ng/mL for MCC-DM1 and 2.00–1000 ng/mL for Lys-MCC-DM1. Intra- and inter-day precision of low, middle and high quality controls was <15%, and accuracy was 99.2–110.9%. The methods were successfully applied to evaluate three catabolites of novel ADCs with N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate linker in vitro and in vivo studies.